Abstract

Background and Aims: MicroRNAs (miRNAs) are short non-coding RNA whose expression levels have diagnostic and prognostic implications in cancer. However, the relevance of aberrantly expressed miRNAs to breast tumour-immune response has not been established. We aimed to assess the association between deregulated miRNAs and tumour immune response in breast molecular cancer subtypes.

Methods: One hundred and seventy ductal infiltrating breast carcinomas (BC) (ER+/HER2− n=85, HER2+ n=40, ER−/HER2− n=45) were evaluated for the presence of cellular immune components by IHC assay. CD3, CD20 were used as markers for T lymphocytes infiltration (TIL), and B lymphocytes infiltration (BIL), respectively, whereas CD68 for the presence of tumour-associated macrophage (TAM). Total RNA (including miRNA) was extracted from all tumours and 32 highly specific miRNAs and 276 immune genes were evaluated by array and qRT-PCR assays. We derived an integrated immune miRNA-regulatory gene profile to tease out relationships of these genes as potential targets of specific miRNAs. In vitro experiments on BC cell lines were performed to confirm potential links between deregulated miRNAs and immune genes.

Results: Lymphocytes infiltration (LI) was most common in HER2+ and ER−/HER2− BC samples (P < .004 and < .001) although B and T cells components were differently distributed in these 2 BC groups. TAM was a frequent event in ER−/HER2− BC subtype (P < .005). By gene and integration analyses we identified important miRNA immune-modulated target genes associated with prognostic factors and with molecular BC subtypes. We focused our studies on miR-181 and miR-135a that were significantly associated with ER−/HER2− (P < .001) and HER2+ BC (P < .002), respectively. BCs with abnormal expression of miR-181 and miR-135a showed an important association with distinct TIL and TAM components and specific immune genes. Importantly, preclinical data on BC cell lines demonstrated that miR-181 and miR-135a were able to regulate important cell immune-modulators such as SEMA3B and STAT6 signalling pathways, respectively. These 2 cellular pathways in turn were capable to regulate specific cascades of immune gene networks.

Conclusion: We identified specific miRNAs associated with immune cellular components and immune-related tumour response in the 3 main molecular BC subtypes (ER+/HER2−, HER2+ and ER−/HER2). Aberrant expression of miR-181 and miR-135a can contribute to BC tumour immune response programs by regulating important gene targets such as SEMA3B and STAT6 signalling pathways in ER−/HER2− and HER2+ tumours, respectively. Modulation of miRNAs activating tumour immune response programs may have important implications for novel therapeutic strategies in BC.